【作者】 吴晓金；

【导师】 吴小平；

【作者基本信息】 福建农林大学 ， 微生物学， 2008， 硕士

【摘要】 从木霉污染的食用菌菌筒和子实体中分离纯化了49株木霉菌株。采用形态学方法以及ITS/5.8S测序分析,对这些木霉进行了分类鉴定。结果表明,中国福建、浙江等省食用菌栽培相关木霉以哈茨木霉(Trichoderma harzianum)和长枝木霉(T.longibrachiaturn)为主,少量为深绿木霉(T.atroviride)和棘孢木霉(T.asperellure);木霉的种类与采集地点、受污染食用菌的种类有一定相关,如在浙江庆元香菇菌筒中分离的木霉主要是哈茨木霉,在广州刺芹侧耳(杏孢菇)菌筒中分离的木霉主要是棘孢木霉,而从福建浦城香菇菌筒中分离的木霉主要是深绿木霉。哈茨木霉、长枝木霉、深绿木霉和棘孢木霉的ITS总长分别为:575 bp-578 bp、579 bp-583 bp、565 bp -567 bp、和560 bp -561 bp,GC含量分别为:55.6%-56.7%、57.2%-58%、55.6%-56.1%和55.8%-55.9%,充分体现了木霉属ITS的长度多态性;ITS1序列总长分别为:221bp-233bp、248 bp-255 bp、214 bp-216 bp和213 bp-214 bp,GC含量分别为:55.4%-57.4%、57.5%-58.7%、55.4%-55.6%和55.6%-55.9%;ITS2序列总长分别为:185bp-188 bp、168 bp-183bp、189 bp-192 bp和188 bp,GC含量分别为:63.1%-65.6%、66.1%-67.9%、62.9%-64.7%和63.8%;所有木霉5.8S完全相同,总长为:159 bp,GC含量为:46.5%。ITS序列最适合用作分类鉴定标记,ITS1不适合用作长枝木霉的分类鉴定,ITS2则种间差异较小的木霉不能得到很好的区分。不同菌株的木霉其RAPD条带,生长最适温度、pH值、培养基含水量都有所不同,显示了食用菌栽培相关木霉具有多样性。木霉对5种食用菌栽培常用消毒剂敏感性测定,显示在同等条件下多菌灵和甲基托布津对木霉抑制效果最好,百菌清效果次之,之后是大生,甲霜灵不太适宜在食用菌栽培中当作防霉剂使用。本文还研究了49株木霉对食用菌菌丝的侵染能力,发现不同木霉的侵染能力不同,同时还发现,对食用菌侵染能力强的木霉对植物病原真菌的侵染能力并不一定强,表现出木霉对真菌侵染的特异性。本文还探讨了木霉侵染食用菌的机理,结果显示,木霉可通过溶壁、缠绕等方式作用于食用菌菌丝细胞壁,木霉发酵液对食用菌和植物病原菌都有很好的抑制作用。木霉几丁质酶活性与侵染性没有直接关联:木霉侵染性强的菌株几丁质酶活性不一定高,产酶活性高的菌株侵染能并不一定强。几丁质酶活性、几丁质酶基因序列及氨基酸序列均与木霉的种类无关,如:同为深绿木霉的66和69,几丁质酶活性分别为:0.0204U/mL.min和0.0044U/mL.min,前者约是后者的5倍;不同种类的木霉可以有相似的几丁质酶序列。几丁质酶活性与侵染性无关,如木霉52无论在植物病原菌还是在食用菌中侵染性都很强,但其几丁质酶活性最弱。木霉几丁质酶基因序列和氨基酸序列与侵染性有一定关系,侵染性强的棘孢木霉29和深绿木霉52同为一类;侵染能力弱的棘孢木霉30和长枝木霉33同聚一类;侵染能力中的哈茨木霉45单独聚为一类;深绿木霉66侵染能力强也单独聚为一类。木霉纤维素酶活性与侵染性没有直接关联。侵染性强的菌株有木霉4、29、52和66,侵染性中的菌株有木霉19、38、63和69,侵染性弱的菌株有木霉28、30、37和70,但产纤维素酶活性强的菌株有木霉29和63,产酶活性中等的菌株有木霉19、28、30、37、38、52、66、69和70,产酶活性弱的菌株有木霉4号。纤维素酶活性、纤维素酶基因序列和氨基酸序列均与木霉的种类无关。克隆得到的5个木霉纤维素酶基因序列中,4株为哈茨木霉,一株为长枝木霉,其基因、cDNA和氨基酸序列同源性均很高,在97.3%～100%之间。木霉纤维素酶基因序列和氨基酸序列与侵染性无关。侵染性弱的哈茨木霉20与侵染性中的哈茨木霉90同源性最近,聚为一类;同一侵染能力的木霉44、47、65和90没有聚在一起。更多还原

【Abstract】 In this paper. 49 strains of Trichoderma spp. which associate with green mold epidemic of edible fungi were researched. Those were isolated from pollution of the edible fungus and fruiting body. This article also classified and identified those strains by morphological and molecular method which was ITS sequence. The results showed that there were mainly Trichoderma harzianum and Trichoderma longibrachiatum in Fujian and Zhejiang provinces and so on in Chinese Mushroom farm. There were also a few Trichoderma atroviride and Trichoderma asperellum. Trichoderma species related to locations and types of edible fungi collected to certain extent, such as there were mainly T.harzianum in Zhejiang Qingyuan Lentinus edodes. There were mainly T.asperellum in Guangzhou Pleurotus Eryngium. But isolation of Lentinus edodes polluted from Fujian Pucheng were mainly T.atroviride. Molecular characteristic of 29 T.harzianum strains, 15 Trichoderma longibrachiatum strains, 3 T.atroviride strains and 2 T.asperellum strains were set out in this article. ITS were 575bp-578bp, 579bp- 583bp, 565bp-567bp and 560bp-561bp, respectively. GC content were 55.6%-56.7% ,57.2%-58%, 55.6%-56.1% and 55.8%-55.9%, respectively. which displayed the ITS length diversity of T.spp. ITS1 sequences were respectively 221bp-233bp, 248bp-255 bp, 214bp-216bp and 213bp-214bp, GC content were 55.4%-57.4 %, 57.5%-58.7% ,55.4% -55.6% and 55.6%-55.9%. ITS2 sequence were 185bp-188bp, 168bp-183bp, 189bp-192bp and 188bp, respectively. GC content were 63.1% -65.6%, 66.1%-67.9%, 62.9%-64.7% and 63.8%, respectively. All of T.spp.’s 5.8 S were 159 bps, GC content were 46.5%. ITS sequences were suitable for classification and identification marker. ITS1 sequence were not suitable. ITS2 can not be a good marker if there were homologous T.spp.. Different strains were different from each other in RAPD and biological characteristic, which showed that there were broadly diversity in T. spp. Sensitivity of Trichoderma to five kinds of fungicide which were usually used in Edible Fungi cultivation were set out in this article. T. spp. can not grow in the thiophanate-methyl and carbendazim PDA culture medium in any concentration. Chlorothalonil and Mancozeb can also restrain Trichoderma growth to some extent. But metalaxyl is not suitable for preventing Trichoderma. Results showed that different T.spp. strain had different aggressive ability and the aggression of Trichoderma toward mushroom was different from it toward Pathogens of plants. That is, the most aggressive strains of T. spp. toward mushroom maybe feebleness toward pathogens of plant, which demonstrated that the specialty of aggressive ability of Trichoderma spp. In this paper the aggressive mechanism of T. spp was also discussed, and the result showed that Trichoderma can via lying cell walls, Winding means etc. to kill edible fungi mycelia. Diffusible inhibitory substances from T.spp. inhibited mycelium growth of edible fungi and pathogen fungi of plant. These T. spp. produced chitinase, but there was no consistent association between levels of chitinase and aggressiveness. T.spp’s chitinase activity were not directly related to their aggressiveness. Aggressive strongly Trichoderma strains which activity of chitinase were not necessarily high, vice versa. There were not consistent association between Chitinase activity and chitinase gene sequence and the amino acid sequence. such as T.atroviride strains 66 and 69 chitinase activity were respectively 0.0204 U / mL.min and 0.0044 U / mL.min. Different species of T. spp. can have similar chitinase sequence. Chitinase activity had nothing to do with the infestation. such as T. atroviride 52 were aggressive strongly both in plant pathogens and the edible fungus. But its chitinase activity was one of the weakest. T.spp chitinase gene and the amino acid sequence had something to do with the infection. Aggressive strong T. atroviride strain 52 and T. asperellum strain 29 were in the same clustering, so and weak aggressive T. asperellum 30 and T. longibrachiatum 33. But the common aggressive T.harzianum 45 and strong aggressive T. atroviride 66 were clustering alone. Trichoderma cellulase were not directly related to infection. Infection strong in edible fungi were Trichoderma strains 4,29,52 and 66, the common were Trichoderma strains 19,38,63 and 69, the weak were Trichoderma strains 28,30,37 and 70. But which produced cellulase in high level were Trichoderma strains 29 and 63. The middle are 19,28,30,37,38,52,66,69 and 70, the weak were T.spp. strains 4. Cellulase activity and cellulase gene sequence and amino acid sequences were unrelated to Trichoderma species. Four of the five cloned Trichoderma cellulase gene sequence were T.harzianum and one of them was T. longibrachiatum. Their gene, cDNA and amino acid sequence were high homology, which can be reached 97.3% to 100%. Cellulase gene sequence and amino acid sequence had nothing to do with the infection. The weak infection of T. harzianum strains 20 and the common strain 90 were homologous recently and clustered together. Ability of the same infection T.spp strains 44,47,65 and 90 did not come together.更多还原